Editing Peptide Conjugation to KLH (section)

== Coupling Peptides to KLH or BSA ==

m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is water insoluble and sensitive to moisture, stored at -20 oC, soluble in DMF (Dimethylformamide), DMSO (Dimethylsulfoxide) its NHS ester and maleimide reactive groups react with amino and sulfhydryl moites, respectively.

'''http://excellgen.net/eln/images/e/ec/Mbs_linker.png'''

1. Dissolve <s>5 mg</s> of KLH or BSA in<s>0.5</s> mL of 0.01 M phosphate buffer (pH 7).

25 mgKLH--> 2.5 ml Buffer

2. Dissolve<s>3 mg</s> of MBS in<s>200 µL</s> DMF.&nbsp; 30 mg-->2 ml

3. <s>Add 70 µL of MBS solution to 0.5 mL of KLH solution.</s> Add 700 µl dropwise, or 10&nbsp;µl&nbsp; while mixing

4. After stirring or rotating for 30 min at room temperature, the KLH/MBS or BSA/MBS solution is passed through a PD-10 column using 0.05 M phosphate buffer (pH 6). Collect the 3.5 mL of purified KLH/MBS or BSA/MBS. Add 0.5 mL of water.

5mg/4 ml, ~ 1mg/ml

5. Dissolve 5 mg of peptide in 100 µL of DMF. Rapidly add 1 mL of purified KLH/MBS or BSA/MBS. Shake rapidly and immediately add 11 µL of 2 N NaOH.

5 mg peptide to ~1 mg&nbsp;activated&nbsp;KLH

6. Check the pH with pH paper. It should be 7.0–7.2. Too high pH or too low pH will stop the reaction between KLH/MBS or BSA/MBS and peptide. If needed, add immediately an appropriate amount of 0.5 N HCl or 2 N NaOH to change the pH.

7. Stir or rotate the solution 3 h or overnight at 4°C. Finally, add 3 mL of ammonium bicarbonate (0.1 M) before lyophilizing the reaction solution

This type of conjugation requires either a N- or C-terminal cysteine.

A total of 3 mg m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is dissolved in 200 ul dimethyl formamide (DMF).

Next, 70 ul MBS/DMF is added to a KLH solution consisting of 5 mg KLH in 0.5 ml 10 mM phosphate buffer, pH 7.0, and it is stirred gently at room temperature for 30 min.

The free crosslinker is removed using a Sephadex G25 size exclusion column. First, the column is equilibrated in 50 mM phosphate buffer (pH 6.0). The KLH reaction mixture is then loaded, and eluted using 50 mM phosphate buffer (pH 6.0). Collect the 3.5 mL of purified KLH/MBS or BSA/MBS. Add 0.5 mL of water.



Conjugation to Peptides



'''Protocol 1'''

A total of 5 mg synthetic peptide is dissolved in 100 μL of DMF. Rapidly add 1 mL of purified KLH/MBS or BSA/MBS. Shake rapidly and immediately add 11 μL of 2 N NaOH to adjust the pH between 7.0-7.2. The reaction will stop if the pH is not in the right range.

The mixture is stirred for 3 hours at room temperature or overnight at 4°C.

Add 3 mL of ammonium bicarbonate (0.1 M) before Lyophilization.

'''Protocol 2'''



1. Reconstitute one vial of the maleimide-activated protein by adding 200 ul of ultrapure water to make a 10 mg/ml solution.

Note: Vortex mixing or heating will cause the protein to precipitate.

2. Dissolve the sulfhydryl-containing hapten in a volume of Conjugation Buffer equal to 1.0-2.5 times the volume of reconstituted activated protein. For example dissolve 2 mg of peptide in 200-500 ul of buffer and add it to the reconstituted protein. Alternatively, if the peptide is freely soluble it may be added as a solid to the protein solution.

Note: For haptens with limited solubility, '''DMSO''' may be used for solubilization. Use approximately '''30'''% DMSO in the final conjugation solution or the carrier protein may irreversibly denature.

3. Immediately mix the peptide and activated protein and react for 2 hours at room temperature.

4. Purify the conjugate by desalting or dialysis to remove EDTA and sodium azide.






'''Note:'''

1. DMF was used as the solvent to solubilize peptides instead of PBS or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7).

2. The last desalting or dialyzing step to remove uncoupled peptides in the traditional method was eliminated.

3. Three milliliters of 0.1 M ammonium bicarbonate was added to the carrier protein conjugated peptide solution before lyophilization, which helped the lyophilization process.

A. Checking Peptide Thiol Groups (see Hint #2)

1. Weigh out about 1 mg of peptide of interest into a tared 50 ml polypropylene conical tube (see Hint #3).

2. Add 0.5 ml of Ellman's Reagent. The solution should turn bright yellow.

3. Dilute the mixture 1:50 in 0.1 M Sodium Phosphate Buffer. Read the absorbance at the 412 nm (Abs412) wavelength, comparing the reading to the Abs412 of the reagent alone diluted to the same concentration.

4. Calculate the apparent molecular weight of the peptide based on thiol groups, using a molar extinction coefficient (εmax) of 14,000. Compare this to the expected molecular weight of the peptide. They should agree within a factor of three, with the estimated molecular weight usually higher (see Hint #4).